Primary cilium assembly or ciliogenesis is a cell cycle-dependent process that occurs at the distal end of the mother centriole. Cells form cilia in interphase or Go and resorb cilia prior to mitosis. Mechanistically ciliogenesis is a poorly understood process. It has been proposed that during ciliogenesis a membrane bilayer forms around the developing microtubule-based axoneme. As the organelle develops it is transported to the cell surface where the ciliary membrane fuses with the plasma membrane. It is generally thought that the ciliary membrane remains separated from the cell surface membrane, thus forming a discrete signaling compartment. We and others have shown that Rab11 and Rab8 members of the Rab small GTPase family, are involved in membrane trafficking processes during ciliary membrane assembly. Rab11 transports Rabin8, a guanine nucleotide exchange factor (GEF), to the centrosome where it activates Rab8-dependent membrane transport important for ciliary membrane assembly. The molecular mechanisms involved in this trafficking pathway are relatively uncharacterized. These and other Rabs may also be important for ciliary signaling. In particular Rab23, a protein mutated in the ciliopathy Capenter Syndrome, has been genetically linked to the sonic hedgehog pathway. During this last fiscal year, I established a laboratory at the NCI. One of the directions of the lab is to study the molecular mechanism of the Rab11-Rab8 ciliary membrane assembly pathway and the role of membrane trafficking in ciliary receptor signaling. We are developing novel advanced high-resolution microscopy techniques to study the dynamics of these transport events. We purchased and implemented a specialized spinning disk confocal microscope to enable simultaneous multi-color live imaging to investigate the dynamics of membrane trafficking processes occurring during ciliary membrane assembly and ciliary receptor/cargo transport. We have successfully used this system to examine primary cilium assembly and ciliary transport. Moreover, were have combined this technology with electron microscopy to examine the higher structure of the primary cilium in a technique known as correlative light and electron microscopy (CLEM). To further study the molecular mechanism of these pathways we have developed biochemistry applications, including tools to isolate protein complexes from cell lines for comparative and quantitative proteomics studies using mass spectrometry. With this approach we have identified Rabin8 interacting proteins important for ciliary assembly including FASN, a lipid modifying enzyme that may be important for ciliary membrane formation.